Having completed the cDNA cloning of the mRNA encoding the myosin heavy chain (MHC) A isoform in chicken intestinal epithelial cells, we initiated studies to complete the nucleotide sequence of the MHC-B cDNA. Since the mRNA encoding MHC-B is predominant in the brain, we cloned the cDNA encoding MHC-B from a chicken brain library. We determined the complete amino acid sequence of a MHC-B (1976 amino acids) and this sequence shows overall similarity to other MHCs, especially to nonsarcomeric type MHCs (MHC-A, B, smooth muscle MHCs). There are 8 regions (5 in the head and 3 in the rod) that show marked identity with respect to amino acid sequence when chicken and Drosophila nonmuscle MHCs are compared. The short uncoiled region at the carboxy-terminus of the molecule has a putative, casein kinase C III phosphorylatable serine residue. The protein kinase C site of MHC-A in the rod region (serine residue) is substituted with a threonine residue in MHC-B. We found one interesting clone which contained a 63 nucleotide insertion at nucleotide 1864 from the first ATG. These nucleotides encode a 21 amino acid insertion at the junction of the 50K and 20K domain in the myosin head region. The mRNA containing this insertion is expressed in the tissues of the central nervous system,. especially cerebellum and brain stem portion. MHC-B' (which contains the insertion) might have some special function in the tissue of the central nervous system. The insertion may alter the properties of myosin. For example, it might affect the actin-activated ATPase activity or the affinity of myosin for actin, because this insertion is located near the actin-binding site in the myosin molecule.